Commercial Polymerases to Create Receptors, Ligands, and Catalysts on Demand Firebird Biomolecular Sciences LLC University of Texas Steven A. Benner Andrew D. Ellington ABSTRACT Innovators at Firebird Biomolecular Sciences LLC have created several generations of an ?artificially ex- panded genetic information system? (AEGIS) that adds eight nucleobases that form four additional pairs (Z:P, S:B, K:X, V:J) to the four natural nucleotides that form the standard G:C and A:T pairs. AEGIS components carry functional groups not found in natural DNA and RNA (collectively xNA) (-NH2, -SH, -COOH), as well as some not found in any natural biopolymer (e.g. ?B(OH)2 and -ONH2). Thus, AEGIS is a new kind of polymer, replicable, evolvable, and adaptable (like DNA) but also having much of the functional potential of proteins. In DNA, AEGIS has been remarkably successful. Six-letter GACTSB AEGIS DNA enables two FDA-approved products that measure hepatitis and HIV viral loads; here the ability of AEGIS DNA to pair only with AEGIS DNA, and not to background xNA ?orthogonality?, allowed these products to earn ca. $100 million each year in their ten year life. The orthogonality of GACTZP AEGIS DNA, as well as its ability to be replicated with high fidelity by DNA polymerases, are today exploited in diagnostics and surveillance for respiratory disease viruses (common and exotic), arboviruses (in mosquitoes), noroviruses, and coronaviruses (e.g. SARS, MERS). Firebird now sees the opportunity to expand its product portfolio from AEGIS DNA to include AEGIS RNA. Its scientists recently reported large-scale syntheses of AEGIS RNA triphosphates and phosphoramidites; these are now sold in its 2016 catalog. Further, Firebird scientists have shown that T7 RNA polymerase makes AEGIS RNA from AEGIS DNA, although inefficiently, and found reverse transcriptases that make AEGIS DNA from AEGIS RNA, in both six and eight letter versions (e.g. GACTSBZP). These are ?leads? for the proposed research. RNA is well-known to have a richer set of folds and more intrinsic potential to serve as receptors and ligands (aptamers, a term and technology invented by the subcontractor). Functionalized AEGIS RNA will thus likely be better than AEGIS DNA in these biomolecules. Functionalized AEGIS RNA will also have enhanced value in laboratory in vitro evolution (LIVE) experiments to create receptors, ligands, and catalysts on demand. Should this vision be realized, it will create a revolutionary transformation of diagnostics and therapeutics. This vision requires better RNA polymerases and reverse transcriptases. While Firebird could undertake their development in-house, advances in the Ellington laboratory at the University of Texas suggests that collabora- tion would achieve this goal faster. Ellington has already created directed evolution platforms to give T7 RNA polymerases and thermostable reverse transcriptases that accept unnatural (but not AEGIS) nucleotides. This collaboration will apply those platforms to create enzymes that interconvert AEGIS DNA and AEGIS RNA. The enzymes themselves will be commercial products; they will also, however, support LIVE with this evolvable functionalized AEGIS biopolymer. Phase 1 will show the feasibility of using Ellington?s platforms to create these enzymes. Given feasibility, the platforms will be applied in Phase 2 to complete 12 letter AEGIS alphabets.